The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.

Summary:Peripheral stem cells were mobilized and collected in 26 pediatric patients with malignant diseases. A total of 47 leukaphereses were performed in the 26 patients. The mean number of nucleated cells collected was 4.5 ؎ 2.6 × 10 8 /kg and the number of CD34 ؉ progenitors collected was 6.7 ؎ 6.8 × 10 6 /kg. CD34-positive selection was performed using a two-step method of magnetic-activated cell sorting (MACS) in 24 patients or a combination of an immunoaffinity column and MACS in two patients. The purity of the positively selected CD34؉ progenitors was 98.8 ؎ 0.7% and the number of isolated CD34 ؉ cells was 6.5 ؎ 5.9 × 10 6 /kg. Thus, the mean recovery of CD34 ؉ cells was 93 ؎ 10%. In 22 of the 26 patients, high-dose chemotherapy was performed with subsequent reinfusion of the highly purified CD34 ؉ cells. In all 22 patients, a normal hematopoietic reconstitution was seen with a mean time of 12.4 ؎ 2.7 days to reach Ͼ0.5 × 10 9 /l neutrophils (range 8-19 days). The time to reach independence from platelet transfusion was 31.6 ؎ 17.0 days (range 16-78 days). There were no transplant-related deaths. In summary, we have shown that mobilized peripheral CD34 ؉ progenitors can be highly purified with a good recovery and that reinfusion of these cells after high-dose chemotherapy results in a rapid, complete and sustained engraftment. We conclude that this method can be used for purging in any CD34-negative malignancies and for autologous T and B cell depletion in the treatment of autoimmune diseases with high-dose immunoablative therapy.

Summary:We present our experience with three clinical scale isolation methods for positive selection of CD34 + progenitors from peripheral blood for autologous and allogeneic transplantation in children. A combination of the CellPro device and the Magnetic Activated Cell Sorting system (MACS), as well as two different combinations of MACS systems were used (VarioMACS-SuperMACS and SuperMACS-SuperMACS). With the CellPro-MACS combination (16 separations), a median purity of 96.2% and a median recovery of 42% CD34 + cells could be achieved, whereas the two step MACS systems (55 and 29 separations) showed a median purity of 97.6% and 98.0% and a median recovery of 96.5% and 97%, respectively. Depletion of T cells was profound (4-5 log). A total of 34 patients in the autologous and 18 patients in the allogeneic setting have been transplanted with highly enriched CD34 + cells, obtained by these methods. Only one patient failed to engraft, all other patients showed a rapid and sustained hematological engraftment with the longest follow-up of 3 years. In summary, especially the two step MACS systems have proven to be appropriate tools for enrichment of CD34 + cells, yielding both high purity and good recovery, and can thus be used for tumor cell purging in the autologous setting and for effective T cell depletion in the allogeneic setting.

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